This invention relates to genetic engineering of plants. More particularly, the invention provides DNA sequences and constructs that are useful to control expression of recombinant genes in plants. Specific constructs of the invention use novel regulatory sequences derived from a maize root preferential cationic peroxidase gene.
Through the use of recombinant DNA technology and genetic engineering, it has become possible to introduce desired DNA sequences into plant cells to allow for the expression of proteins of interest. However, obtaining desired levels of expression remains a challenge. To express agronomically important genes in crops at desired levels through genetic engineering requires the ability to control the regulatory mechanisms governing expression in plants, and this requires access to suitable regulatory sequences that can be coupled with the genes it is desired to express.
A given project may require use of several different expression elements, for example one set to drive a selectable marker or reporter gene and another to drive the gene of interest. The selectable marker may not require the same expression level or pattern as that required for the gene of interest. Depending upon the particular project, there may be a need for constitutive expression, which directs transcription in most or all tissues at all times, or there may be a need for tissue specific expression. For example, a root specific or root preferential expression in maize would be highly desirable for use in expressing a protein toxic to pests that attack the roots of maize.
Cells use a number of regulatory mechanisms to control which genes are expressed and the level at which they are expressed. Regulation can be transcriptional or post-transcriptional and can include, for example, mechanisms to enhance, limit, or prevent transcription of the DNA, as well as mechanisms that limit the life span of the mRNA after it is produced. The DNA sequences involved in these regulatory processes can be located upstream, downstream or even internally to the structural DNA sequences encoding the protein product of a gene.
Initiation of transcription of a gene is regulated by a sequence, called the promoter, located upstream (5xe2x80x2) of the coding sequence. Eukaryotic promoters generally contain a sequence with homology to the consensus 5xe2x80x2-TATAAT-3xe2x80x2 (TATA box) about 10-35 base pairs (bp) upstream of the transcription start (CAP) site. Most maize genes have a TATA box 29 to 34 base pairs upstream of the CAP site. In most instances the TATA box is required for accurate transcription initiation. Further upstream, often between xe2x88x9280 and xe2x88x92100, there can be a promoter element with homology to the consensus sequence CCAAT. This sequence is not well conserved in many species including maize. However, genes which have this sequence appear to be efficiently expressed. In plants the CCAAT xe2x80x9cboxxe2x80x9d is sometimes replaced by the AGGA xe2x80x9cboxxe2x80x9d. Other sequences conferring tissue specificity, response to environmental signals or maximum efficiency of transcription may be found interspersed with these promoter elements or found further in the 5xe2x80x2 direction from the CAP site. Such sequences are found within 400 bp of the CAP site, but may extend as far as 1000 bp or more.
Promoters can be classified into two general categories. xe2x80x9cConstitutivexe2x80x9d promoters are expressed in most tissues most of the time. Expression from a constitutive promoter is more or less at a steady state level throughout development. Genes encoding proteins with housekeeping functions are often driven by constitutive promoters. Examples of constitutively expressed genes in maize include actin and ubiquitin. Wilmink et al. (1995). xe2x80x9cRegulatedxe2x80x9d promoters are typically expressed in only certain tissue types (tissue specific promoters) or at certain times during development (temporal promoters). Examples of tissue specific genes in maize include the zeins (Kriz et al., (1987)) which are abundant storage proteins found only in the endosperm of seed. Many genes in maize are regulated by promoters that are both tissue specific and temporal.
It has been demonstrated that promoters can be used to control expression of foreign genes in transgenic plants in a manner similar to the expression pattern of the gene from which the promoter was originally derived. The most thoroughly characterized promoter tested with recombinant genes in plants has been the 35S promoter from the Cauliflower Mosaic Virus (CaMV) and its derivatives. U.S. Pat. No. 5,352,065; Wilmink et al. (1995); Datla et al. (1993). Elegant studies conducted by Benfey et al. (1984) reveal that the CaMV 35S promoter is modular in nature with regards to binding to transcription activators. U.S. Pat. No. 5,097,025; Benfey et al. (1989) and (1990). Two independent domains result in the transcriptional activation that has been described by many as constitutive. The 35S promoter is very efficiently expressed in most dicots and is moderately expressed in monocots. The addition of enhancer elements to this promoter has increased expression levels in maize and other monocots. Constitutive promoters of monocot origin (that are not as well studied) include the polyubiquitin-1 promoter and the rice actin-1 promoter. Wilmink et al. (1995). In addition, a recombinant promoter, Emu, has been constructed and shown to drive expression in monocots in a constitutive manner, Wilmink et al. (1995).
Few tissue specific promoters have been characterized in maize. The promoters from the zein gene and oleosin gene have been found to regulate GUS in a tissue specific manner. Kriz et al. (1987); Lee and Huang (1994). No root specific promoters from maize have been described in the literature. However, promoters of this type have been characterized in other plant species.
Despite both the important role of tissue specific promoters in plant development, and the opportunity that availability of a root preferential promoter would represent for plant biotechnology, relatively little work has yet been done on the regulation of gene expression in roots. Yamamoto reported the expression of E. coli: uidA gene, encoding xcex2-glucuronidase (GUS), under control of the promoter of a tobacco (N. tabacum) root-specific gene, TobRB7. Yamamoto et al. (1991), Conkling et al. (1990). Root specific expression of the fusion genes was analyzed in transgenic tobacco. Significant expression was found in the root-tip meristem and vascular bundle. EPO Application Number 452 269 (De Framond) teaches that promoters from metallathionein-like genes are able to function as promoters of tissue-preferential transcription of associated DNA sequences in plants, particularly in the roots. Specifically, a promoter from a metallathionein-like gene was operably linked to a GUS reporter gene and tobacco leaf disks were transformed. The promoter was shown to express in roots, leaves and stems. WO 9113992 (Croy, et al.) teaches that rape (Brassica napus L.) extensin gene promoters are capable of directing tissue-preferential transcription of associated DNA sequences in plants, particularly in the roots. Specifically, a rape extensin gene promoter was operably linked to a extA (extensin structural gene) and tobacco leaf disks were transformed. It was reported that northern analysis revealed no hybridization of an extensin probe to leaf RNA from either control or transformed tobacco plants and hybridization of the extensin probe to transgenic root RNA of all transformants tested, although the levels of hybridization varied for the transformants tested. While each of these promoters has shown some level of tissue-preferential gene expression in a dicot model system (tobacco), the specificity of these promoters, and expression patterns and levels resulting from activity of the promoters, has yet to be achieved in monocots, particularly maize.
DNA sequences called enhancer sequences have been identified which have been shown to enhance gene expression when placed proximal to the promoter. Such sequences have been identified from viral, bacterial, and plant gene sources. An example of a well characterized enhancer sequence is the ocs sequence from the octopine synthase gene in Agrobacterium tumefaciens. This short (40 bp) sequence has been shown to increase gene expression in both dicots and monocots, including maize, by significant levels. Tandem repeats of this enhancer have been shown to increase expression of the GUS gene eight-fold in maize. It remains unclear how these enhancer sequences function. Presumably enhancers bind activator proteins and thereby facilitate the binding of RNA polymerase II to the TATA box. Grunstein (1992). WO95/14098 describes testing of various multiple combinations of the ocs enhancer and the mas (mannopine synthase) enhancer which resulted in several hundred fold increase in gene expression of the GUS gene in transgenic tobacco callus.
The 5xe2x80x2 untranslated leader sequence of mRNA, introns, and the 3xe2x80x2 untranslated region of mRNA affect expression by their effect on post-transcription events, for example by facilitating translation or stabilizing mRNA.
Expression of heterologous plant genes has also been improved by optimization of the non-translated leader sequence, i.e. the 5xe2x80x2 end of the mRNA extending from the 5xe2x80x2 CAP site to the AUG translation initiation codon of the mRNA. The leader plays a critical role in translation initiation and in regulation of gene expression. For most eukaryotic mRNAs, translation initiates with the binding of the CAP binding protein to the mRNA CAP. This is then followed by the binding of several other translation factors, as well as the 43S ribosome pre-initiation complex. This complex travels down the mRNA molecule while scanning for an AUG initiation codon in an appropriate sequence context. Once this has been found, and with the addition of the 60S ribosomal subunit, the complete 80S initiation complex initiates protein translation. Pain (1986); Kozak (1986). Optimization of the leader sequence for binding to the ribosome complex has been shown to increase gene expression as a direct result of improved translation initiation efficiency. Significant increases in gene expression have been produced by addition of leader sequences from plant viruses or heat shock genes. Raju et al. (1993); Austin (1994) reported that the length of the 5xe2x80x2 non-translated leader was important for gene expression in protoplasts.
In addition to the untranslated leader sequence, the region directly around the AUG start appears to play an important role in translation initiation. Luerhsen and Walbot (1994). Optimization of the 9 bases around the AUG start site to a Kozak consensus sequence was reported to improve transient gene expression 10-fold in BMS protoplasts. McElroy et al. (1994).
Studies characterizing the role of introns in the regulation of gene expression have shown that the first intron of the maize alcohol dehydrogenase gene (Adh-I) has the ability to increase expression under anaerobiosis. Callis et al. (1987). The intron also stimulates expression (to a lesser degree) in the absence of anaerobiosis. This enhancement is thought to be a result of a stabilization of the pre-mRNA in the nucleus. Mascarenhas et al. reported a 12-fold and 20-fold enhancement of CAT expression by use of the Adh-I intron. Mascarenhas et al. (1990). Several other introns have been identified from maize and other monocots which increase gene expression. Vain et al. (1996).
The 3xe2x80x2 end of the mRNA can also have a large effect on expression, and is believed to interact with the 5xe2x80x2 CAP. Sullivan (1993). The 3xe2x80x2untranslated region (3xe2x80x2UTR) has been shown to have a significant role in gene expression of several maize genes. Specifically, a 200 base pair 3xe2x80x2 sequence has been shown to be responsible for suppression of light induction of the maize small m3 subunit of the ribulose-1,5-biphosphate carboxylase gene (rbc/m3) in mesophyll cells. Viret et al. (1994). Some 3xe2x80x2UTRs have been shown to contain elements that appear to be involved in instability of the transcript. Sullivan et al. (1993). The 3xe2x80x2UTRs of most eukaryotic genes contain consensus sequences for polyadenylation. In plants, especially maize, this sequence is not very well conserved. The 3xe2x80x2untranslated region, including a polyadenylation signal, derived from a nopaline synthase gene (3xe2x80x2 nos) is frequently used in plant genetic engineering. Few examples of heterologous 3xe2x80x2UTR testing in maize have been published.
Important aspects of the present invention are based on the discovery that DNA sequences derived from a maize root specific cationic peroxidase gene are exceptionally useful for use in regulating expression of recombinant genes in plants.
The peroxidases (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) are highly catalytic enzymes with many potential substrates in the plant. See Gaspar, et al. (1982). They have been implicated in such diverse functions as secondary cell wall biosynthesis, wound-healing, auxin catabolism, and defense of plants against pathogen attack. See Lagrimini and Rothstein (1987); Morgens et al. (1990); Nakamura et al. (1988); Fujiyama et al. (1988); and Mazza et al. (1980).
Most higher plants possess a number of different peroxidase isozymes whose pattern of expression is tissue specific, developmentally regulated, and influenced by environmental factors. Lagrimini and Rothstein (1987). Based upon their isoelectric point, plant peroxidases are subdivided into three subgroups: anionic, moderately anionic, and cationic.
The function of anionic peroxidase isozymes (pI, 3.5-4.0) is best understood. Isozymes from this group are usually cell wall associated. They display a high activity for polymerization of cinnamyl alcohols in vitro and have been shown to function in lignification and cross-linking of extensin monomers and feruloylated polysaccharides. Lagrimini and Rothstein (1987). In both potato and tomato, expression of anionic peroxidases have been shown to be induced upon both wound induction and abscisic acid treatment. Buffard et al. (1990). This suggests their involvement in both wound healing and in the regulation of tissue suberization.
Moderately anionic peroxidase isozymes (pI, 4.5-6.5) are also cell wall associated and have some activity toward lignin precursors. In tobacco, isozymes of this class have been shown to be highly expressed in wounded stem tissue Fujiyama et al. (1988). These isozymes may also serve a function in suberization and wound healing. Morgens et al. (1990).
The actual function of cationic peroxidase isozymes (pI, 8.1-11) in the plant remains unclear. Some members of this group, however, have been shown to efficiently catalyze the synthesis of H2O2 from NADH and H2O. Others are localized to the central vacuole. In the absence of H2O2, some of these isozymes possess indoleacetic acid oxidase activity. Lagrimini and Rothstein (1987).
Electrophoretic studies of maize peroxidases have revealed 13 major isozymes. Brewbaker et al. (1985). All isozymes were judged to be functional as monomers, despite major differences in molecular weight. All maize tissues had more than one active peroxidase locus, and all loci were tissue-specific. The peroxidases have proved unique in that no maize tissue has been found without activity, and no peroxidase has proven expressed in all maize tissues.
The invention provides isolated DNA molecules derived from the per5 maize root preferential cationic peroxidase gene that can be used in recombinant constructs to control expression of genes in plants. More particularly, the invention provides isolated DNA molecules derived from the per5 promoter sequence and having as at least a part of its sequence bp 4086-4148 of SEQ ID NO 1. Preferred embodiments are isolated DNA molecules that have as part of their sequences bp 4086 to 4200, bp 4086 to 4215, bp 3187 to 4148, bp 3187 to 4200, bp 3187 to 4215, bp 2532-4148, bp 2532 to 4200, bp 2532 to 4215, bp 1-4148, bp, bp 1-4200, or bp 1-4215 of SEQ ID NO 1.
The invention also provides isolated DNA molecules selected from the following per5 intron sequences: bp 4426-5058, bp 4420-5064, bp 5251-5382, bp 5245-5388, bp 5549-5649, and bp 5542-5654 of SEQ ID NO 1.
The invention also provides isolated DNA molecules derived from the per5 transcription termination sequence and having the sequence of bp 6068-6431 of SEQ ID NO 1.
In another of its aspects, the present invention provides a recombinant gene cassette competent for effecting preferential expression of a gene of interest in a selected tissue of transformed maize, said gene cassette comprising:
a) a promoter from a first maize gene, said first maize gene being one that is naturally expressed preferentially in the selected tissue;
b) an untranslated leader sequence;
c) the gene of interest, said gene being one other than said first maize gene;
d) a 3xe2x80x2UTR;
said promoter, untranslated sequence, gene of interest, and 3xe2x80x2UTR being operably linked from 5xe2x80x2 to 3xe2x80x2; and
e) an intron sequence that is incorporated in said untranslated leader sequence or in said gene of interest, said intron sequence being from an intron of a maize gene that is preferentially expressed in said selected tissue.
A related embodiment of the invention is a recombinant gene cassette competent for effecting constitutive expression of a gene of interest in transformed maize comprising:
a) a promoter from a first maize gene, said first maize gene being one that is naturally expressed preferentially in a specific tissue;
b) an untranslated leader sequence;
c) the gene of interest, said gene being one other than said first maize gene;
d) a 3xe2x80x2UTR;
said promoter, untranslated sequence, gene of interest, and 3xe2x80x2UTR being operably linked from 5xe2x80x2 to 3xe2x80x2; and
e) an intron sequence that is incorporated in said untranslated leader or in said gene of interest, said intron sequence being from an intron of a maize gene that is naturally expressed constitutively.
In a particular embodiment the intron is one from the maize AdhI expressed gene, and the resulting recombinant gene cassette provides constitutive expression in maize.
In another of its aspects, the invention provides DNA constructs comprising, operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction,
a) a promoter having as at least part of its sequence bp 4086-4148 bp of SEQ ID NO 1;
b) an untranslated leader sequence comprising bp 4149-4200 of SEQ ID NO 1,
c) a gene of interest not naturally associated with said promoter, and
d) a 3xe2x80x2UTR.
Preferred embodiments of this aspect of the invention are those wherein the promoter comprises bp 3187 to 4148, bp 2532-4148, or bp 1-4148 of SEQ ID NO 1. Particularly preferred are each of the preferred embodiments wherein said 3xe2x80x2UTR has the sequence of bp 6066-6340 or bp 6066-6439 of SEQ ID NO 1.
In another of its aspects, the invention provides DNA constructs comprising, operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction,
a) a promoter having as at least part of its sequence bp 4086-4148 bp of SEQ ID NO 1;
b) an untranslated leader sequence not naturally associated with said promoter,
c) a gene of interest,
d) a 3xe2x80x2UTR.
Preferred embodiments of this aspect of the invention are those wherein the promoter comprises bp 3187 to 4148, bp 2532-4148, or bp 1-4148 of SEQ ID NO 1. Particularly preferred are each of the preferred embodiments wherein said 3xe2x80x2UTR has the sequence of bp 6066-6340 or bp 6066-6439 of SEQ ID NO 1.
In another of its aspects, the invention provides a DNA construct comprising, operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction,
a) a promoter having as at least part of its sequence bp 4086-4148 bp of SEQ ID NO 1;
b) an untranslated leader sequence comprising bp 4149-4200 of SEQ ID NO 1;
c) an intron selected from the group consisting of an AdhI gene intron and bp 4426-5058 of SEQ ID NO 1;
d) a gene of interest; and
e) a 3xe2x80x2UTR.
Preferred embodiments of this aspect of the invention are again those wherein the promoter comprises bp 3187 to 4148, bp 2532-4148, or bp 1-4148 of SEQ ID NO 1. Particularly preferred are each of the preferred embodiments wherein said 3xe2x80x2UTR has the sequence of bp 6066-6340 or bp 6066-6439 of SEQ ID NO 1.
In another of its aspects, the invention provides a DNA construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction,
a) a promoter having as at least part of its sequence bp 4086-4148 bp of SEQ ID NO 1;
b) an untranslated leader sequence;
c) an intron selected from the group consisting of an AdhI gene intron and bp 4426-5058 of SEQ ID NO 1;
d) a cloning site;
e) a 3xe2x80x2UTR.
In accordance with another significant aspect of the invention, there is provided a recombinant gene cassette comprised of the following operably linked sequences, from 5xe2x80x2 to 3xe2x80x2: a promoter; an untranslated leader sequence; a gene of interest; and the per5 3xe2x80x2UTR, bp 6068-6431 of SEQ ID NO 1.
In another of its aspects, the invention provides a plasmid comprising a promoter having as at least part of its sequence bp 4086-4148 of SEQ ID NO 1.
In another of its aspects, the invention provides a transformed plant comprising at least one plant cell that contains a DNA construct of the invention. The plant may be a monocot or dicot. Preferred plants are maize, rice, cotton and tobacco.
In another of its aspects, the invention provides seed or grain that contains a DNA construct of the invention.
In one of its aspects, the present invention relates to regulatory sequences derived from the maize root preferential cationic peroxidase protein (per5) that are able to regulate expression of associated DNA sequences in plants. More specifically, the invention provides novel promoter sequences and constructs using them. It also provides novel DNA constructs utilizing the per5 untranslated leader and/or 3xe2x80x2UTR. It also provides novel DNA constructs utilizing the introns from the per5 gene.
The DNA sequence for a 6550 bp fragment of the genomic clone of the maize root-preferential cationic peroxidase gene is given in SEQ ID NO 1. The sequence includes a 5xe2x80x2 flanking region (nt 1-4200), of which nucleotides 4149-4200 correspond to the untranslated leader sequence. The coding sequence for the maize root-preferential cationic peroxidase is composed of four exons: exon 1 (nt 4201-4425), exon 2 (nt 5059-5250), exon 3 (nt 5383-5547), and exon 4 (nt 5649-6065). It should be noted that the first 96 nucleotides of exon 1 (nt 4201-4296) code for a 32 amino acid signal peptide, which is excised from the polypeptide after translation to provide the mature protein. Three introns were found: intron 1 (nt 4426-5058), intron 2 (5251-5382), and intron 3 (5548-5648). The 3xe2x80x2 flanking region (373 nucleotides in length) extends from nucleotide 6069 (after the UGA codon at nucleotides 6066-6068) to nucleotide 6550, including a polyadenylation signal at nucleotides 6307-6312.
We have discovered that promoters derived from certain tissue preferential maize genes require the presence of an intron in the transcribed portion of the gene in order for them to provide effective expression in maize and that the temporal and tissue specificity observed depends on the intron used. A recombinant gene cassette having a tissue preferential maize promoter, but lacking an intron in the transcribed portion of the gene, does not give appropriate expression in transformed maize. If the transcribed portion of the cassette includes an intron derived from a maize gene of similar tissue specificity to the maize gene from which the promoter was obtained, the gene cassette, will restore tissue preferential expression in maize. The intron may be, but need not necessarily be, from the same gene as the promoter. If an intron derived from another maize gene, such as AdhI intron 1, is used in a gene cassette with a promoter from a tissue preferential maize gene, the cassette will give generally constitutive expression in maize. We have also found that these considerations apply to transgenic maize, but not to transgenic rice. Tissue preferential maize promoters can be used to drive recombinant genes in rice without an intron.
In accordance with the foregoing unexpected and significant findings, the present invention provides a recombinant gene cassette competent for effecting preferential expression of a gene of interest in a selected tissue of transformed maize, said gene cassette comprising:
a) a promoter from a first maize gene, said first maize gene being one that is naturally expressed preferentially in the selected tissue;
b) an untranslated leader sequence;
c) the gene of interest, said gene being one other than said first maize gene;
d) a 3xe2x80x2UTR;
said promoter, untranslated sequence, gene of interest, and 3xe2x80x2UTR being operably linked from 5xe2x80x2 to 3xe2x80x2; and
e) an intron sequence that is incorporated in said untranslated leader sequence or in said gene of interest, said intron sequence being from an intron of a maize gene that is preferentially expressed in said selected tissue.
The promoter used in this embodiment can be from any maize gene that is preferentially expressed in the tissue of interest. Such maize genes can be identified by conventional methods, for example, by techniques involving differential screening of mRNA sequences.
A detailed example of identification and isolation of a tissue preferential maize gene is given herein for the root preferential maize cationic peroxidase gene. The method illustrated in this example can be used to isolate additional genes from various maize tissues.
Examples of tissue preferential maize genes that have promoters suitable for use in the invention include: O-methyl transferase and glutamine synthetase 1.
A preferred promoter is the per5 promoter, i.e. the promoter from the root preferential maize cationic peroxidase gene. Particularly preferred is the promoter comprising bp 1 to 4215 of SEQ ID NO 1.
The non-translated leader sequence can be derived from any suitable source and may be specifically modified to increase the translation of the mRNA. The 5xe2x80x2 non-translated region may be obtained from the promoter selected to express the gene, the native leader sequence of the gene or coding region to be expressed, viral RNAs, suitable eukaryotic genes, or may be a synthetic sequence.
The gene of interest may be any gene that it is desired to express in plants. Particularly useful genes are those that confer tolerance to herbicides, insects, or viruses, and genes that provide improved nutritional value or processing characteristics of the plant. Examples of suitable agronomically useful genes include the insecticidal gene from Bacillus thuringiensis for conferring insect resistance and the 5xe2x80x2-enolpyruvyl-3xe2x80x2-phosphoshikimate synthase (EPSPS) gene and any variant thereof for conferring tolerance to glyphosate herbicides. Other suitable genes are identified hereinafter. As is readily understood by those skilled in the art, any agronomically important gene conferring a desired trait can be used.
The 3xe2x80x2UTR, or 3xe2x80x2 untranslated region, that is employed is one that confers efficient processing of the mRNA, maintains stability of the message and directs the addition of adenosine ribonucleotides to the 3xe2x80x2 end of the transcribed mRNA sequence. The 3xe2x80x2UTR may be native with the promoter region, native with the structural gene, or may be derived from another source. Suitable 3xe2x80x2UTRs include but are, not limited to: the per5 3xe2x80x2UTR, and the 3xe2x80x2UTR of the nopaline synthase (nos) gene.
The intron used will depend on the particular tissue in which it is desired to preferentially express the gene of interest. For tissue preferential expression in maize, the intron should be selected from a maize gene that is naturally expressed preferentially in the selected tissue.
The intron must be incorporated into a transcribed region of the cassette. It is preferably incorporated into the untranslated leader 5xe2x80x2 of the gene of interest and 3xe2x80x2 of the promoter or within the translated region of the gene.
Why certain tissue preferential maize genes require an intron to enable effective expression in maize tissues is not known, but experiments indicate that the critical event is post-transcriptional processing. Accordingly, the present invention requires that the intron be provided in a transcribed portion of the gene cassette.
A related embodiment of the invention is a recombinant gene cassette competent for effecting constitutive expression of a gene of interest in transformed maize comprising:
a) a promoter from a first maize gene, said first maize gene being one that is naturally expressed preferentially in a specific tissue;
b) an untranslated leader sequence;
c) the gene of interest, said gene being one other than said first maize gene;
d) a 3xe2x80x2UTR;
said promoter, untranslated sequence, gene of interest, and 3xe2x80x2UTR being operably linked from 5xe2x80x2 to 3xe2x80x2; and
e) an intron sequence that is incorporated in said untranslated leader or in said gene of interest, said intron sequence being from an intron of a maize gene that is naturally expressed constitutively.
This embodiment differs from the previous embodiments in that the intron is one from a gene expressed in most tissues, and the expression obtained from the resulting recombinant gene cassette in maize is constitutive. Suitable introns for use in this embodiment of the invention include AdhI intron 1, Ubiquitin intron 1, and Bronze 2 intron 1. Particularly preferred is the AdhI intron 1. Although it has previously been reported that the AdhI intron 1 is able to enhance expression of constitutively expressed genes, it has never been reported or suggested that the AdhI intron can alter the tissue preferential characteristics of a tissue preferential maize promoter.
The present invention is generally applicable to the expression of structural genes in both monocotyledonous and dicotyledonous plants. This invention is particularly suitable for any member of the monocotyledonous (monocot) plant family including, but not limited to, maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, and dates. A preferred application of the invention is in production of transgenic maize plants.
This invention, utilizing a promoter constructed for monocots, is particularly applicable to the family Graminaceae, in particular to maize, wheat, rice, oat, barley and sorghum.
In accordance with another aspect of the invention, there is provided a recombinant gene cassette comprised of: a promoter; an untranslated leader sequence; a gene of interest; and the per5 3xe2x80x2UTR. Use of the per5 3xe2x80x2UTR provides enhanced expression compared to similar gene cassettes utilizing the nos 3xe2x80x2UTR.
The promoter used with the per5 3xe2x80x2UTR can be any promoter suitable for use in plants. Suitable promoters can be obtained from a variety of sources, such as plants or plant DNA viruses. Preferred promoters are the per5 promoter, the 35T promoter (described hereinafter in Examples 20 and 23), and the ubiquitin promoter. Useful promoters include those isolated from the caulimovirus group, such as the cauliflower mosaic virus 19S and 35S (CaMV19S and CaMV35S) transcript promoters. Other useful promoters include the enhanced CaMV35S promoter (eCaMV35S) as described by Kat et al. (1987) and the small subunit promoter of ribulose 1,5-bisphosphate carboxylase oxygenase (RUBISCO). Examples of other suitable promoters are rice actin gene promoter; cyclophilin promoter; AdhI gene promoter, Callis et al. (1987); Class I patatin promoter, Bevan et al. (1986); ADP glucose pyrophosphorylase promoter; .beta.-conglycinin promoter, Tierney et al. (1987); E8 promoter, Deikman et al. (1988); 2AII promoter, Pear et al. (1989); acid chitinase promoter, Samac et al. (1990). The promoter selected should be capable of causing sufficient expression of the desired protein alone, but especially when used with the per5 3xe2x80x2UTR, to result in the production of an effective amount of the desired protein to cause the plant cells and plants regenerated therefrom to exhibit the properties which are phenotypically caused by the expressed protein.
The untranslated leader used with the per5 3xe2x80x2UTR is not critical. The untranslated leader will typically be one that is naturally associated with the promoter. The untranslated leader may be one that has been modified in accordance with another aspect of the present invention to include an intron. It may also be a heterologous sequence, such as one provided by U.S. Pat. No. 5,362,865. This non-translated leader sequence can be derived from any suitable source and can be specifically modified to increase translation of the mRNA.
The gene of interest may be any gene that it is desired to express in plants, as described above.
The terms xe2x80x9cper5 3xe2x80x2UTRxe2x80x9d and/or xe2x80x9cper5 transcription termination regionxe2x80x9d are intended to refer to a sequence comprising bp 6068 to 6431 of SEQ ID NO 1.
Construction of gene cassettes utilizing the per5 3xe2x80x2UTR is readily accomplished utilizing well known methods, such as those disclosed in Sambrook et al. (1989); and Ausubel et al. (1987).
As used in the present application, the terms xe2x80x9croot-preferential promoterxe2x80x9d, xe2x80x9croot-preferential expressionxe2x80x9d, xe2x80x9ctissue-preferential expressionxe2x80x9d and xe2x80x9cpreferential expressionxe2x80x9d are used to indicate that a given DNA sequence derived from the 5xe2x80x2 flanking or upstream region of a plant gene of which the structural gene is expressed in the root tissue exclusively, or almost exclusively and not in the majority of other plant parts. This DNA sequence when connected to an open reading frame of a gene for a protein of known or unknown function causes some differential effect; i.e., that the transcription of the associated DNA sequences or the expression of a gene product is greater in some tissue, for example, the roots of a plant, than in some or all other tissues of the plant, for example, the seed. Expression of the product of the associated gene is indicated by any conventional RNA, cDNA, protein assay or biological assay, or that a given DNA sequence will demonstrate.
This invention involves the construction of a recombinant DNA construct combining DNA sequences from the promoter of a maize root-preferential cationic peroxidase gene, a plant expressible structural gene (e.g. the GUS gene (Jefferson, (1987)) and a suitable terminator.
The present invention also includes DNA sequences having substantial sequence homology with the specifically disclosed regulatory sequences, such that they are able to have the disclosed effect on expression.
As used in the present application, the term xe2x80x9csubstantial sequence homologyxe2x80x9d is used to indicate that a nucleotide sequence (in the case of DNA or RNA) or an amino acid sequence (in the case of a protein or polypeptide) exhibits substantial, functional or structural equivalence with another nucleotide or amino acid sequence. Any functional or structural differences between sequences having substantial sequence homology will be de minimis; that is they will not affect the ability of the sequence to function as indicated in the present application. For example, a sequence which has substantial sequence homology with a DNA sequence disclosed to be a root-preferential promoter will be able to direct the root-preferential expression of an associated DNA sequence. Sequences that have substantial sequence homology with the sequences disclosed herein are usually variants of the disclosed sequence, such as mutations, but may also be synthetic sequences.
In most cases, sequences having 95% homology to the sequences specifically disclosed herein will function as equivalents; and in many cases considerably less homology, for example 75% or 80%, will be acceptable. Locating the parts of these sequences that are not critical may be time consuming, but is routine and well within the skill in the art.
DNA encoding the maize root-preferential cationic peroxidase promoter may be prepared from chromosomal DNA or DNA of synthetic origin by using well-known techniques. Specifically comprehended as part of this invention are genomic DNA sequences. Genomic DNA may be isolated by standard techniques. Sambrook et al. (1989); Mullis et al. (1987); Horton et al. (1989); Erlich (ed.)(1989). It is also possible to prepare synthetic sequences by oligonucleotide synthesis. See Caruthers (1983) and Beaucage et al. (1981).
It is contemplated that sequences corresponding to the above noted sequences may contain one or more modifications in the sequences from the wild-type but will still render the respective elements comparable with respect to the teachings of this invention. For example, as noted above, fragments may be used. One may incorporate modifications into the isolated sequences including the addition, deletion, or nonconservative substitution of a limited number of various nucleotides or the conservative substitution of many nucleotides. Further, the construction of such DNA molecules can employ sources which have been shown to confer enhancement of expression of heterologous genes placed under their regulatory control. Exemplary techniques for modifying oligonucleotide sequences include using polynucleotide-mediated, site-directed mutagenesis. See Zoller et al. (1984); Higuchi et al. (1988); Ho et al. (1989); Horton et al. (1989); and PCR Technology: Principles and Applications for DNA Amplification, (ed.) Erlich (1989).
In one embodiment, an expression cassette of this invention, will comprise, in the 5xe2x80x2 to 3xe2x80x2 direction, the maize root-preferential cationic peroxidase promoter sequence, in reading frame, one or more nucleic acid sequences of interest followed by a transcript termination sequence. The expression cassette may be used in a variety of ways, including for example, insertion into a plant cell for the expression of the nucleic acid sequence of interest.
The tissue-preferential promoter DNA sequences are preferably linked operably to a coding DNA sequence, for example, a DNA sequence which is transcribed into RNA, or which is ultimately expressed in the production of a protein product.
A promoter DNA sequence is said to be xe2x80x9coperably linkedxe2x80x9d to a coding DNA sequence if the two are situated such that the promoter DNA sequence influences the transcription of the coding DNA sequence. For example, if the coding DNA sequence codes for the production of a protein, the promoter DNA sequence would be operably linked to the coding DNA sequence if the promoter DNA sequence affects the expression of the protein product from the coding DNA sequence. For example, in a DNA sequence comprising a promoter DNA sequence physically attached to a coding DNA sequence in the same chimeric construct, the two sequences are likely to be operably linked.
The DNA sequence associated with the regulatory or promoter DNA sequence may be heterologous or homologous, that is, the inserted genes may be from a plant of a different species than the recipient plant. In either case, the DNA sequences, vectors and plants of the present invention are useful for directing transcription of the associated DNA sequence so that the mRNA transcribed or the protein encoded by the associated DNA sequence is expressed in greater abundance in some plant tissue, such as the root, leaves or stems, than in the seed. Thus, the associated DNA sequence preferably may code for a protein that is desired to be expressed in a plant only in preferred tissue, such as the roots, leaves or stems, and not in the seed.
Promoters are positioned 5xe2x80x2 (upstream) to the genes that they control. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art and demonstrated herein with multiple copies of regulatory elements, some variation in this distance can occur.
Any plant-expressible structural gene can be used in these constructions. A structural gene is that portion of a gene comprising a DNA segment encoding a protein, polypeptide, antisense RNA or ribozyme or a portion thereof. The term can refer to copies of a structural gene naturally found within the cell, but artificially introduced, or the structural gene may encode a protein not normally found in the plant cell into which the gene is introduced, in which case it is termed a heterologous gene.
The associated DNA sequence may code, for example, for proteins known to inhibit insects or plant pathogens such as fungi, bacteria and nematodes. These proteins include, but are not limited to, plant non-specific lipid acyl hydrolases, especially patatin; midgut-effective plant cystatins, especially potato papain inhibitor; magainins, Zasloff (1987); cecropins, Hultmark et al. (1982); attacins, Hultmark et al. (1983); melittin; gramicidin S, Katsu et al. (1988); sodium channel proteins and synthetic fragments, Oiki et al. (1988); the alpha toxin of Staphylococcus aureus, Tobkes et al. (1985); apolipoproteins and fragments thereof, Knott et al. (1985) and Nakagawa et al. (1985); alamethicin and a variety of synthetic amphipathic peptides, Kaiser et al. (1987); lectins, Lis et al. (1986) and Van Parijs et al. (1991); pathogenesis-related proteins, Linthorst (1991); osmotins and permatins, Vigers et al. (1992) and Woloscuk et al. (1991); chitinases; glucanases, Lewah et al. (1991); thionins, Bohlmann and Apel (1991); protease inhibitors, Ryan (1990); plant anti-microbial peptides, Cammue et al. (1992); and polypeptides from Bacillus thuringiensis, which are postulated to generate small pores in the insect gut cell membrane, Knowles et al. (1987) and Hofte and Whitely (1989).
The structural gene sequence will generally be one which originates from a plant of a species different from that of the target organism. However, the present invention also contemplates the root preferential expression of structural genes which originates from a plant of the same species as that of the target plant but which are not natively expressed under control of the native root preferential cationic peroxidase (per5) promoter.
The structural gene may be derived in whole or in part from a bacterial genome or episome, eukaryotic genomic, mitochondrial or plastid DNA, cDNA, viral DNA, or chemically synthesized DNA. It is possible that a structural gene may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, rearrangements and substitutions of one or more nucleotides. The structural gene may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate plant-functional splice junctions. The structural gene may be a composite of segments derived from a plurality of sources, naturally occurring or synthetic. The structural gene may also encode a fusion protein, so long as the experimental manipulations maintain functionality in the joining of the coding sequences.
The use of a signal sequence to secrete or sequester in a selected organelle allows the protein to be in a metabolically inert location until released in the gut environment of an insect pathogen. Moreover, some proteins are accumulated to higher levels in transgenic plants when they are secreted from the cells, rather than stored in the cytosol. Hiatt, et al. (1989).
At the 3xe2x80x2 terminus of the structural gene will be provided a termination sequence which is functional in plants. A wide variety of termination regions are available that may be obtained from genes capable of expression in plant hosts, e.g., bacterial, opine, viral, and plant genes. Suitable 3xe2x80x2UTRs include those that are known to those skilled in the art, such as the nos 3xe2x80x2, tmL 3xe2x80x2, or acp 3xe2x80x2, for example.
In preparing the constructs of this invention, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Adapters or linkers may be employed for joining the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
In carrying out the various steps, cloning is employed, so as to amplify a vector containing the promoter/gene of interest for subsequent introduction into the desired host cells. A wide variety of cloning vectors are available, where the cloning vector includes a replication system functional in E. coli and a marker which allows for selection of the transformed cells. Illustrative vectors include pBR322, pUC series, pACYC184, Bluescript series (Stratagene) etc. Thus, the sequence may be inserted into the vector at an appropriate restriction site(s), the resulting plasmid used to transform the E. coli host (e.g., E. coli strains HB101, JM101 and DH5xcex1), the E. coli grown in an appropriate nutrient medium and the cells harvested and lysed and the plasmid recovered. Analysis may involve sequence analysis, restriction analysis, electrophoresis, or the like. After each manipulation the DNA sequence to be used in the final construct may be restricted and joined to the next sequence, where each of the partial constructs may be cloned in the same or different plasmids.
Vectors are available or can be readily prepared for transformation of plant cells. In general, plasmid or viral vectors should contain all the DNA control sequences necessary for both maintenance and expression of a heterologous DNA sequence in a given host. Such control sequences generally include, in addition to the maize root-preferential cationic peroxidase promoter sequence (including a transcriptional start site), a leader sequence and a DNA sequence coding for translation start-signal codon (generally obtained from either the maize root-preferential cationic peroxidase gene or from the gene of interest to be expressed by the promoter or from a leader from a third gene which is known to work well or enhance expression in the selected host cell), a translation terminator codon, and a DNA sequence coding for a 3xe2x80x2 non-translated region containing signals controlling messenger RNA processing. Selection of appropriate elements to optimize expression in any particular species is a matter of ordinary skill in the art utilizing the teachings of this disclosure; in some cases hybrid constructions are preferred, combining promoter elements upstream of the tissue preferential promoter TATA and CAAT box to a minimal 35S derived promoter consisting of the 35S TATA and CAAT box. Finally, the vectors should desirably have a marker gene that is capable of providing a phenotypical property which allows for identification of host cells containing the vector, and an intron in the 5xe2x80x2 untranslated region, e.g., intron 1 from the maize alcohol dehydrogenase gene that enhances the steady state levels of mRNA of the marker gene.
The activity of the foreign gene inserted into plant cells is dependent upon the influence of endogenous plant DNA adjacent the insert. Generally, the insertion of heterologous genes appears to be random using any transformation technique; however, technology currently exists for producing plants with site specific recombination of DNA into plant cells (see WO/9109957). The particular methods used to transform such plant cells are not critical to this invention, nor are subsequent steps, such as regeneration of such plant cells, as necessary. Any method or combination of methods resulting in the expression of the desired sequence or sequences under the control of the promoter is acceptable.
Conventional technologies for introducing biological material into host cells include electroporation, as disclosed in Shigekawa and Dower (1988), Miller, et al. (1988), and Powell, et al (1988); direct DNA uptake mechanisms, as disclosed in Mandel and Higa (1972) and Dityatkin, et al. (1972), Wigler, et al. (1979) and Uchimiya, et al. (1982); fusion mechanisms, as disclosed in Uchidaz, et al. (1980); infectious agents, as disclosed in Fraley, et al. (1986) and Anderson (1984); microinjection mechanisms, as disclosed in Crossway, et al. (1986); and high velocity projectile mechanisms, as disclosed in EPO 0 405 696.
Plant cells from monocotyledonous or dicotyledonous plants can be transformed according to the present invention. Monocotyledonous species include barley, wheat, maize, oat and sorghum and rice. Dicotyledonous species include tobacco, tomato, sunflower, cotton, sugarbeet, potato, lettuce, melon, soybean and canola (rapeseed).
The appropriate procedure to transform a selected host cell may be chosen in accordance with the host cell used. Based on the experience to date, there appears to be little difference in the expression of genes, once inserted into cells, attributable to the method of transformation itself. Once introduced into the plant tissue, the expression of the structural gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome.
Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. The appropriate procedure to produce mature transgenic plants may be chosen in accordance with the plant species used. Regeneration varies from species to species of plants. Efficient regeneration will depend upon the medium, on the genotype and on the history of the culture. Once whole plants have been obtained, they can be sexually or clonally reproduced in such a manner that at least one copy of the sequence is present in the cells of the progeny of the reproduction. Seed from the regenerated plants can be collected for future use, and plants grown from this seed. Procedures for transferring the introduced gene from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.